Calcium-binding proteins: differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF-hand calcium-binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium-binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin-immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin-immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium-binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal.     

Calcium‐binding proteins: Differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF‐hand calcium‐binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium‐binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin‐immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin‐immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium‐binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 207–217, 1999     

Distribution of GluR1 is altered in the olfactory bulb following neonatal naris occlusion

The olfactory system is well suited for studies of glutamate receptor plasticity. The sensory neurons are glutamatergic, and they turn over throughout life, and the olfactory bulb neurons that process their inputs express many of the known glutamate receptor subunits. Neonatal naris occlusion alters olfactory bulb development and the expression of certain neuroactive substances and receptors, at least in part due to loss of the sensory inputs. We therefore postulated that neonatal naris occlusion might alter glutamate receptor expression during postnatal development. Single nares of newborn mice were occluded on postnatal days 1-2, and the distribution of glutamate receptor subunits was evaluated using immunoperoxidase methods. Light microscopic examination on postnatal day 6 failed to reveal adult-like staining of neuronal cell bodies in the olfactory bulbs. By day 12, cell bodies that were immunoreactive (-IR) for the GluR1 subunit were visible in the external plexiform layer (EPL) of both sides. By day 18, many of the GluR1-IR cell bodies could be identified as cell types that had previously been reported to express homomeric GluR1 receptors. Analysis of single, mid-dorsal sections from 18-25-day-old mice showed that the medial EPL of the occluded side had a significantly lower density of these cell bodies. The GluR1 staining of the adjacent mitral cell layer (MCL) was also heavier on the occluded side, but no gross differences in staining for other glutamate receptor subunits were observed. Neonatal naris occlusion therefore appears to provide a new model for studying expression of GluR1 receptors during the development of a discrete population of olfactory bulb neurons.     

Structure of the olfactory bulb in tadpoles of Xenopus laevis

The structure of the olfactory bulb in tadpoles of Xenopus laevis (stages 54-56) was studied using axon tracing (with biocytin or low-weight dextran) and immunocytochemical techniques. Filling the olfactory nerve with biocytin made the nerve layer and the glomeruli visible. Dye injections into the glomerular layer labeled the lateral olfactory tract. Vice versa, dye injections into the lateral olfactory tract made mitral cells and their glomerular branching patterns visible. Anti-GABA antiserum stained periglomerular and granule cells, while the olfactory nerve and mitral cells were labeled by antiglutamate antiserum. We describe the layering, the numbers of cells and glomeruli, and their localization in both the main and the accessory olfactory bulb.     

The postnatal development of the main olfactory bulb of the rat

The postnatal development from birth to 1 year of the main olfactory bulb was examined quantitatively. The volume of the main olfactory bulb increased over seven-fold by day 30 and remained unchanged thereafter. During the same period the volume of the granular layer increased 18-fold and the mean areas of the olfactory glomeruli increased seven-fold. The mean areas of mitral cell perikarya doubled between the neonatal and juvenile periods. The total number of the mitral cells, however, declined during the first three postnatal weeks. In the internal granular layer of the main olfactory bulb, 89% of the granule cells were acquired postnatally. Much of the cellular gain occurred during the first 3 weeks, with the period of maximum acquisition between days 8 and 14. The number of subependymal cells, the precursors of granule cells, reached a peak at 12 days and gradually declined. But some primitive cells could still be found at one year of age and there was an increase in the total number of granule cells beyond day 30. The mean nuber of internal granular layer cells in a single main olfactory bulb of adult rats was about 5 X 10(6); the number of mitral cells about 4 X 10(4). In the animals injected with 3H-thymidine on day 20 and killed 2 h after injection a small but significant proportion of cells was labelled in the subependymal layer but few in the internal granular layer. In the animals killed 20 and 40 days after injection there was a 10--11-fold rise in the proportion of labelled internal granular layer cells. The proportion of labelled internal granular layer cells decreased in longer survival groups but the total number of labelled cells remained the same, even in year-old animals. However, the total number of internal granular layer cells in the sections examined increased with age.     

Comparison of identified mitral and tufted cells in freely breathing rats: II. Odor-evoked responses

Mitral and tufted cells are the 2 types of output neurons of the main olfactory bulb. They are located in distinct layers, have distinct projection patterns of their dendrites and axons, and likely have distinct relationships with the intrabulbar inhibitory circuits. They could thus be functionally distinct and process different aspects of olfactory information. To examine this possibility, we compared the odor-evoked responses of identified single units recorded in the mitral cell layer (MCL units), in the core of the external plexiform layer (not at the glomerular border tufted cells), or at the glomerular border of this layer (GB tufted cells) of the entire olfactory bulb. Differences between mitral and tufted cells were observed only when subtle aspects of the responses were explored, such as the firing rate per respiratory cycle or the distribution of firing activity along the respiratory cycle. By contrast, more clear differences were found when the 2 subtypes of tufted cells were examined separately. GB units were significantly more responsive, had significantly higher firing activity, and showed greater activity at the transition between inspiration and expiration. The projection-type tufted cells situated closer to the entrance of the olfactory bulb may thus form a distinct physiological class of output neurons and differ from mitral cells and other tufted cells in the manner of processing olfactory information.     

Mitral cell differentiation and synaptogenesis of rat presumptive olfactory bulb in organ culture

Summary The ultrastructure of differentiating rat presumptive olfactory bulb in organ culture was investigated with particular reference to mitral cell differentiation and formation of synapses. The presumptive olfactory bulb and olfactory mucosa were dissected en bloc from rat embryos on the fifteenth day of gestation and cultured for 7 days, after which the expiants were examined by electron microscopy. The presumptive olfactory bulb had differentiated into a laminated structure with layers corresponding to the glomerular, external plexiform and mitral cell layers. Mitral-like cells were identified by their location and large cell size. Ultrastructural observations indicated that they were relatively well-differentiated. Their dendrites extended into the glomerular layer in which they were postsynaptic to incoming olfactory axons. The distal part of these dendrites frequently contained coated vesicles. Both asymmetrical and symmetrical synapses were found. The symmetrical synapses involved dendrodendritic contacts between periglomerular cells. Synapses in reciprocal arrangements were not observed in the organ cultures.     

Effects of unilateral olfactory deprivation in the developing opossum,Monodelphis domestica

Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 429–438, 1997     

GABA immunoreactivity in the main olfactory bulb of the frog Rana temporaria

Immunoreactivity for gamma-aminobutyric acid (GABA) was localized at the light microscopic level in the main olfactory bulb (MOB) of the frog, Rana temporaria. By means of free-floating peroxidase-antiperoxidase immunocytochemical technique, GABA was found in a large number of neurons in the granular cell layer, in a few small somata in the mitral cell layer and in two different types of cell somata in the glomerular layer. Individual GABA-immunopositive cells were found in the olfactory nerve layer. GABA immunostaining was also localized in cell processes and fiber fragments. There were many immunoreactive puncta in all layers of the MOB. GABA-positive punctate structures often outlined immunonegative cells in the mitral cell and glomerular layers. Rounded tightly packed groups of immunoreactive puncta were found only along ventral border of the glomerular layer. The results are discussed in comparison with data obtained on mammalian MOB in terms of MOB functional organization.     

The α6 integrin subunit in the developing mouse olfactory bulb

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the α 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the α 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an α 6 knockout (α 6^?/?). Immunohistochemical analyses and confocal microscopy were used to examine α 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the α 6^?/? mouse. In CD-1 mice from E13 to E17, α 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; ～E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the α 6^?/? mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for α 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.     

棕色田鼠脑和行为不同发育阶段副嗅球和主嗅球的细胞活动

本文运用免疫组化显示Fos蛋白的方法首次研究了棕色田鼠脑和行为不同发育阶段副嗅球和主嗅球的细胞活动。当不同年龄阶段的幼鼠同时暴露于自己家庭的熟悉底物和另一家庭的陌生底物时 ,嗅闻和呆在自己熟悉底物上的时间较多 ,直到产后 15d、 2 0d和 2 5d时 ,幼鼠探究不同底物的行为显示出显著性差异。脑的大小随着日龄增加而增加 ,但从产后 1到 15d ,脑重、脑宽和嗅球大小随着日龄增加特别显著。当不同日龄幼鼠暴露于陌生底物或者暴露于自己的熟悉底物时 ,从产后 5到 15日龄 ,主嗅球僧帽细胞层、颗粒细胞层、副嗅球僧帽细胞层和颗粒细胞层Fos免疫阳性细胞随着日龄明显增加 ,但直到 15和 30日龄时 ,和对照组相比 ,陌生底物可引起幼鼠主嗅球Fos免疫阳性细胞明显增加 ,从 2 0日龄起 ,陌生底物可引起副嗅球Fos免疫阳性细胞明显增加。主嗅球颗粒细胞层Fos免疫阳性细胞随着日龄的增加从边缘到中心逐渐出现 ,而副嗅球Fos免疫阳性细胞随着日龄的增加从顶部到底部逐渐出现。以上结果说明产后第 1d到 15d左右可能是棕色田鼠脑结构发育的重要阶段 ,而从此以后棕色田鼠主嗅球和副嗅球就具有区别熟悉气味和陌生气味的能力 ,表明棕色田鼠行为、脑发育和细胞活动间有紧密关系     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.     

Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.     

Laminar Distribution of Putative Neurotransmitter Amino Acids and Ligand Binding Sites in the Dog Olfactory Bulb

Coronal sections of frozen dog olfactory bulb have been dissected into four anatomically distinct layers. The laminar distribution of ten amino acids, the dipeptide carnosine, and nine [3H]ligand binding sites in these layers was determined. GABA and tyrosine levels were highest in the mitral cell-granule cell layer, and glutamate levels were slightly elevated in the glomerular layer. The distributions of all other amino acids did not show significant differences across the layers. Carnosine was predominantly localized in the fiber and glomerular layers. With the exception of quinuclidinyl benzilate, the [3H]ligand binding sites showed more discrete distributions. Muscimol, diazepam, kainic acid, and spiroperidol binding were predominantly localized in the mitral cell-granule cell layer, where clonidine binding was at a minimum. Dihydromorphine binding was high in both the fiber and the mitral cell-granule cell layers. Carnosine binding was maximal in the glomerular layer. The implications of these observations with regard to biochemical and neurophysiological data are discussed.     

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.     

Ontogenetic development of neural responses in the olfactory bulb of laboratory mice

Summary In laboratory mice (strain NMRI) the ontogenetic development of single unit activity in the olfactory bulb was investigated. From postnatal day 10 on, spontaneously active neurons were recorded with glass-microelectrodes, and their responses to olfactory stimuli were tested (butyric acid, geraniol, grass- and nest-odour).From day 10 to 13 only very few neurons were recordable (and most of these elements were too weak and were lost before being stimulated). At day 14 the number of recordable neurons increased rapidly, and by day 15 spontaneously active neurons reached adult level in terms of incidence and electric properties.There were 3 types of neurons: 1. respiration synchronous elements; 2. bursting neurons not correlated with respiration; 3. continuously, but randomly, firing elements (about 60% of all neurons). Reactions to odour stimuli (excitation, ca. 50%; inhibition, ca. 34%; complex reactions, ca. 12%; change in activity pattern, ca. 4%) occurred as soon as the cells were stable enough for testing. The reaction patterns showed no age specific differences; the duration of the responses varied from 100 ms to 100 s.In younger animals (P11–P14) the percentage of responses was slightly smaller (47%) than in the older ones (P30–P50; 64% response to olfactory stimulation). For some of the odours tested the proportion of responding cells differed depending on age (for instance grass odour evoked a response in 40% of the cells in young ones, but in 65% in adults).Abbreviations AP action potential - In interneuron - MTc mitral or tufted cell - P10 postnatal day 10     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

Analytical processing of binary mixture information by olfactory bulb glomeruli

Odors are rarely composed of a single compound, but rather contain a large and complex variety of chemical components. Often, these mixtures are perceived as having unique qualities that can be quite different than the combination of their components. In many cases, a majority of the components of a mixture cannot be individually identified. This synthetic processing of odor information suggests that individual component representations of the mixture must interact somewhere along the olfactory pathway. The anatomical nature of sensory neuron input into segregated glomeruli with the bulb suggests that initial input of odor information into the bulb is analytic. However, a large network of interneurons within the olfactory bulb could allow for mixture interactions via mechanisms such as lateral inhibition. Currently in mammals, it is unclear if postsynaptic mitral/tufted cell glomerular mixture responses reflect the analytical mixture input, or provide the initial basis for synthetic processing with the olfactory system. To address this, olfactory bulb glomerular binary mixture representations were compared to representations of each component using transgenic mice expressing the calcium indicator G-CaMP2 in olfactory bulb mitral/tufted cells. Overall, dorsal surface mixture representations showed little mixture interaction and often appeared as a simple combination of the component representations. Based on this, it is concluded that dorsal surface glomerular mixture representations remain largely analytical with nearly all component information preserved.